背景:有研究表明微小RNA 及潜在靶基因在肝癌中起重要作用,但具体机制仍不清楚.目的:构建靶向血管内皮生长因子基因微小RNA 真核表达载体,评估其转染人肝癌细胞株HepG2 后血管内皮生长因子基因的干扰效果.方法:根据血管内皮生长因子序列设计合成4 对微小RNA 不同干扰片段,克隆到pcDNA6.2-GW/EmGFP-微小RNA 真核表达载体上,测序分析鉴定插入序列的完整性;并将其转染至HepG-2 细胞株中.采用实时荧光定量聚合酶链式反应分析血管内皮生长因子微小RNA 干扰效果,蛋白印迹技术测定重组体对血管内皮生长因子基因蛋白表达情况.结果与结论:构建的4 组重组体插入片段的碱基序列完全正确,重组体能干扰肝癌细胞HepG2 细胞血管内皮因子基因的表达,4 组重组体基因的mRNA 的表达水平和蛋白表达水平与阴性对照组相比明显降低(P < 0.05),其中血管内皮生长因子微小RNA-3 表达水平最低,抑制率87%.结果证实,实验成功构建血管内皮生长因子微小RNA 表达载体,在体外能有效抑制HepG2 细胞血管内皮生长因子基因表达.%BACKGROUND: Studies have shown that the microRNA (miRNA) and its potential target gene play an important role in hepatocellular carcinoma, but the exact mechanism is still unclear. OBJECTIVE: To construct a vascular endothelial growth factor (VEGF) miRNA expressing eukaryotic vector and to identify biological activity of VEGF miRNA transfected into HepG-2 cells. METHODS: According to the sequence of VEGF mRNA, the VEGF miRNA was designed and synthesized, and then cloned into the pcDNA6.2-GW/EmGFP-miRNA vector and transfected into HepG-2 cell lines. The integrity of the insert fragment was detected using colony PCR and sequencing analysis. The biological activity of VEGF miRNA by way of real-time PCR and Western blot was determined. RESULTS AND CONCLUSION: Sequences of the inset fragment in the four miRNA expressing recombinants were correct. VEGF mRNA expression of the four miRNA recombinants were significantly decreased (P < 0.05), especially in the VEGF-miRNA-3 with an inhibitory rate of 87%. Four VEGF-targeting miRNA expressing recombinants were successfully constructed which can significantly inhibit VEGF gene expression in HepG2 cells.
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